Elevated STAT3 Phosphorylation Research Articles

DDDR-31. TARGETING CANCER STEM CELLS WITH A CDK2 INHIBITOR IN GLIOBLASTOMA

Abstract Glioblastoma (GB) is the most common and aggressive adult brain tumor with no cure. Brain tumor stem cells (BTSCs) are a rare population of self-renewing multipotent stem cells in GB that contribute to tumorigenesis, therapeutic resistance and tumour recurrence. Here, we report a CDK2 inhibitor that suppresses BTSCs via OSM/OSMR/STAT3 signalling pathway. To begin with we performed high throughput screening (HTS) of ~8400 compounds including FDA-approved drugs in patient derived human BTSCs that naturally harbour EGFRvIII mutation and elevated STAT3 phosphorylation, in search of compounds that can suppress EGFRvIII/OSMR/STAT3 oncogenic pathway. The screen led to the identification of a panel of CDK inhibitors that possessed important characteristics including a) ability to cross the blood-brain barrier with mall molecular weights of 277-566 kDa, b) low Topological Polar Surface Area (TPSA) of 76-115 A°, c) a low number of Hydrogen Bond Donors (HBDs) (1-4), and d) cLogP values ranging from 2-4. Following counter screens, we focused on a CDK2 inhibitor that significantly and most efficiently reduced OSM/OSMR signalling and STAT3 activation. Importantly, we found that the compound inhibited the self-renewal and growth of BTSCs in EGFRvIII subtype of BTSCs. This research has led to future investigation on in vivo assessment of this CDK2 inhibitor in combination with ionizing radiation and chemotherapy in preclinical models of GB.

The impact of cellular elements of TME on melanoma biology and its sensitivity to EGFR and MET targeted therapy.

Microenvironment of the melanoma consists of cellular elements like fibroblasts, adipocytes, and keratinocytes as well as extracellular matrix and physicochemical conditions. In our previous research, we have established that melanoma influences strongly above mentioned cells present in the tumor niche and recruits them to support cancer progression. In this work, we evaluated the impact of cancer-associated cells, namely fibroblasts (CAFs), adipocytes (CAAs), and keratinocytes (CAKs) on melanoma proliferation, signaling pathways activation, metabolism as well as the effectiveness of used anti-cancer therapy. Obtained results indicated elevated phosphorylation of STAT3, upregulated GLUT1 and GLUT3 as well as downregulated of MCT-1 expression level in melanoma cells under the influence of all examined cells present in the tumor niche. The proliferation of melanoma cells was increased after co-culture with CAFs and CAKs, while epithelial-mesenchymal transition markers' expression level was raised in the presence of CAFs and CAAs. The level of perilipin 2 and lipid content was elevated in melanoma cells under the influence of CAAs. Moreover, increased expression of CYP1A1, gene encoding drug metabolizing protein, in melanoma cells co-cultured with CAFs and CAKs prompted us to verify the effectiveness of the previously proposed by us anti-melanoma therapy based on combination of EGFR and MET inhibitors. Obtained results indicate that the designed therapy is still efficient, even if the fibroblasts, adipocytes, and keratinocytes, are present in the melanoma vicinity.

TARGETING CANCER STEM CELLS WITH A CDK2 INHIBITOR IN GLIOBLASTOMA

Abstract Glioblastoma (GB) is the most common and aggressive adult brain tumor with no cure. Brain tumor stem cells (BTSCs) are a rare population of self-renewing multipotent stem cells in GB that contribute to tumorigenesis, therapeutic resistance, and tumour recurrence. Here, we report a CDK2 inhibitor that suppresses BTSCs via OSM/OSMR/STAT3 signalling pathway. To begin with, we performed high throughput screening (HTS) of ~8400 compounds including FDA-approved drugs in patient derived human BTSCs that naturally harbour EGFRvIII mutation and elevated STAT3 phosphorylation, in search of compounds that can suppress EGFRvIII/OSMR/STAT3 oncogenic pathway. The screen led to the identification of a panel of CDK inhibitors that possessed important characteristics including a) ability to cross the blood-brain barrier with mall molecular weights of 277-566 kDa, b) low Topological Polar Surface Area (TPSA) of 76-115 A°, c) a low number of Hydrogen Bond Donors (HBDs) (1-4), and d) cLogP values ranging from 2-4. Following counter screens, we focused on a CDK2 inhibitor that significantly and most efficiently reduced OSM/OSMR signalling and STAT3 activation. Importantly, we found that the compound inhibited the self-renewal and growth of BTSCs in EGFRvIII subtype of BTSCs. This research has led to future investigation on in vivo assessment of this CDK2 inhibitor in combination with ionizing radiation and chemotherapy in preclinical models of GB.

STAT3 as a Potential Target for Tumor Suppressive Effects of 15-Deoxy-Δ12,14-prostaglandin J2 in Triple Negative Breast Cancer.

STAT3 plays a prominent role in proliferation and survival of tumor cells. Thus, STAT3 has been considered to be a prime target for development of anti-cancer therapeutics. The electrophilic cyclopentenone prostaglandin,15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been well recognized for its capability to modulate intracellular signaling pathways involved in cancer cell growth and progression. We previously reported that 15d-PGJ2 had potent cytotoxicity against harvey-ras transformed human mammary epithelial cells through direct interaction with STAT3. In this study, we have attempted to verify the inhibitory effects of 15d-PGJ2 on STAT3 signaling in human breast tumor cells. The triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 displaying constitutive phosphorylation of STAT3 on the tyrosine 705 (Tyr705) residue, underwent apoptosis upon inhibition of STAT3 by 15d-PGJ2. In contrast, estrogen receptor positive MCF-7 breast cancer cells that do not exhibit elevated STAT3 phosphorylation were much less susceptible to 15d-PGJ2-induced apoptosis as assessed by PARP cleavage. Furthermore, 15d-PGJ2 inhibited interleukin-6-induced tyrosine phosphorylation of STAT3 in LNCaP cells. According to molecular docking studies, 15d-PGJ2 may preferentially bind to the cysteine 259 residue (Cys259) present in the coiled-coil domain of STAT3. Site-directed mutagenesis of STAT3 identified Cys259 to be the critical amino acid for the 15d-PGJ2-induced apoptosis as well as epithelial-to-mesenchymal transition. Taken together, these findings suggest STAT3 inactivation through direct chemical modification of its Cys259 as a potential therapeutic approach for treatment of triple negative breast cancer treatment.

Oncostatin-M-Reactive Pericytes Aggravate Blood–Brain Barrier Dysfunction by Activating JAK/STAT3 Signaling In Vitro

Oncostatin M (OSM) is a cytokine of the interleukin (IL)-6 family members. It induces blood–brain barrier (BBB) dysfunction by activating Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) 3 pathways in brain endothelial cells. Brain pericytes located around microvessels are one of the BBB constituents. Pericytes work as a boundary surface between the blood circulation and brain parenchyma, and their functions are altered under pathophysiological conditions, leading to BBB dysregulation. However, it remains unknown whether pericytes are associated with OSM-induced BBB dysfunction. We demonstrated that pericyte exposure to OSM (100 ng/mL) elevated phosphorylation of STAT3, a main OSM signaling pathway, and that pericytes expressed OSM receptors (OSMRs) including OSMRβ and glycoprotein 130. These results suggest that pericytes are able to respond to OSM. To determine the effects of OSM-reactive pericytes on BBB functions, rat brain endothelial cell (RBEC) monolayers were cultured with OSM-treated pericytes. The presence of pericytes exposed to 100 ng/mL of OSM for 48 h aggravated both the elevated permeability to sodium fluorescein and the lowered transendothelial electrical resistance which were induced by OSM in RBECs. This OSM-reactive pericyte-induced aggravation of lowered RBEC barrier function was reversed by ruxolitinib, a JAK inhibitor. These findings suggest that activated JAK/STAT3 signaling in pericytes contributes to OSM-produced BBB breakdown. Thus, OSM-reactive pericytes may have to be considered a characteristic machinery in the formation and progression of BBB breakdown under pathological conditions associated with increased OSM levels.

Effects of CKMT1 on radiosensitivity of nasopharyngeal carcinoma cells

Purpose: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear. The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity.Material and methods: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2. Biological changes were detected after cells exposing to different doses of X-ray to determine the role of CKMT1 on NPC cell radiosensitivity.Results: CKMT1 promotes proliferation and migration in NPC cell lines CNE-1 and CNE-2. Overexpression of CKMT1 in CNE-1 cells enhanced colony formation rates, reduced G2/M phase cell cycle arrest, lowered apoptosis rate and c-PARP level, and elevated STAT3 phosphorylation level after radiation treatment. While knocking out CKMT1 using the CRISPR/Cas9 system in CNE-2 cells lowered colony formation rates, increased G2/M phase cell cycle arrest, apoptosis rates, and c-PARP levels, and decreased STAT3 phosphorylation in response to radiation treatment.Conclusions: NPC cells with higher CKMT1 exhibited lower radiosensitivity through promoting phosphorylation of STAT3. Our findings suggest that CKMT1 may be an alternative radiotherapeutic target in NPC therapy.

MBD2-mediated Th17 differentiation in severe asthma is associated with impaired SOCS3 expression

T helper 17 (Th17) cells has proven to be crucial in the pathogenesis of severe asthma. Although it is known that Suppressor of cytokine signaling 3 (SOCS3) is involved in differentiation of Th17 cells but, how it affects severe asthma is uncertain. Since previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) null mice was deficient in Th17 cell differentiation, the aim of the present study was to understand how MBD2 interacts with SOCS3 to regulate Th17 cell differentiation in severe asthma. Here, we show that SOCS3 expression was significantly decreased in Th17-mediated severe asthmatic mice, accompanied by elevated STAT3 phosphorylation and RORγt expression. Knock-down of SOCS3 promoted the differentiation of naïve T cells into Th17 cells through STAT3/RORγt pathway. Meanwhile, MBD2 was overexpressed in Th17-mediated severe asthmatic mice. Intervention of MBD2 expression lead to a negative change of SOCS3 expression, whereas the differentiation of Th17 cells showed positive change. In addition, MBD2 knockout (MBD2-KO) mice displayed increased SOCS3 expression and decreased Th17 differentiation after severe asthma modeling. Taken together, our results suggest that MBD2 might facilitate Th17 cell differentiation via down-regulating SOCS3 expression in severe asthma. These findings uncover new roles for SOCS3 and MBD2, and provide a potential target for treatment of severe asthma.

Aberrant Activation of the PI3K/mTOR Pathway Promotes Resistance to Sorafenib in AML

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant MV4-11 and MOLM-13 cell lines by sustained treatment with the FLT3 inhibitor sorafenib. MV4-11 cells express only FLT3-ITD, while MOLM-13 cells express wild-type FLT3 and FLT3-ITD. Both cell lines are dependent on FLT3 activation as sorafenib, PKC-412, and AC220, but not imatinib, dasatinib, nilotinib or bosutinib, inhibit cell survival in both cell lines. After treatment with sorafenib for 90 days, we observed that both cell lines displayed resistance to sorafenib as well as to AC220 suggesting that sustained treatment with an FLT3 inhibitor results in acquired resistance to multiple FLT3 inhibitors.To test whether sorafenib was still effective in FLT3 inhibition, we treated sensitive and resistant cells with sorafenib or DMSO and then stimulated with FLT3 ligand (FL). While DMSO-treated resistant cells responded to FL as expected, sorafenib-treated cells displayed poor FLT3 activation, suggesting that sorafenib was still capable of inhibiting FLT3 activation in the resistant cells. Surprisingly we observed that the resistant cells treated with DMSO had a much more robust response to ligand in terms of FLT3 activation. Similar results were observed with AKT and ERK activation, as sorafenib-treated cells poorly responded to FL-stimulation. Furthermore, resistant cells treated with sorafenib could still form colonies similar to DMSO-treated cells indicating that these cells were no longer dependent on FLT3 activation, although sorafenib could partially block FLT3 activation.To determine whether secondary mutations occurred in FLT3, we sequenced the whole coding region using Sanger sequencing. Except for a mutation in the extracellular domain in all four cell lines no mutations were detected in the inhibitor binding site. Similar to Sanger sequencing, mass spectroscopic analysis of affinity-enriched FLT3 indicated no differences in the intracellular part of FLT3 between sensitive and resistant cells. Because we observed an unexpected activation of FLT3 in resistant cells stimulated with FL, we hypothesized that certain FLT3 residues remain hyper-tyrosine phosphorylated. To test that we used phospho-specific antibodies against known FLT3 tyrosine phosphorylation sites. Although we observed an increase in total FLT3 tyrosine phosphorylation, we were unable to identify a single site that was selectively hyper-phosphorylated. Instead, all sites remained slightly more phosphorylated compared to control cells.Gene expression analysis of mRNA from sensitive and resistant cell lines using ANOVA and significance analysis of microarrays (SAM) suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype. In addition to pathway enrichment, using a phospho-protein antibody array, we found that phosphorylation of the mTOR substrates S6K and AKT were selectively increased in resistant cells. We also observed an increase in STAT3 phosphorylation. Elevated STAT3 phosphorylation was probably due to the previously described upregulation of JAK3 expression in sorafenib-resistant AML. We also observed enrichment of an mTOR signature in AML blasts from eight patients with sorafenib-resistant AML (GSE35907) and in AML blasts from patients expressing FLT3-ITD compared to those lacking FLT3-ITD (525 samples, GSE14468).Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in the resistant cell lines. Treatment of cells with a higher concentration of gedatolisib did not alter the phosphorylation of other signaling proteins except for AKT and S6K which was detected by a phospho-specific antibody array. The array data was further verified with western blotting using phospho-specific antibodies against AKT, ERK1/2, p38 and S6K. These results suggest that gedatolisib efficiently blocks the downstream effectors PI3K/mTOR without affecting other signaling pathways.Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD dependent AML results in resistance to drugs targeting FLT3 and treatment with selective PI3K/mTOR inhibitors are likely to be effective in overcoming acquired resistance in AML patients. DisclosuresStegmaier:Novartis Pharmaceuticals: Consultancy.

Myeloid-Cell Protein Tyrosine Phosphatase-1B Deficiency in Mice Protects Against High-Fat Diet and Lipopolysaccharide-Induced Inflammation, Hyperinsulinemia, and Endotoxemia Through an IL-10 STAT3-Dependent Mechanism

Protein tyrosine phosphatase-1B (PTP1B) negatively regulates insulin and leptin signaling, rendering it an attractive drug target for treatment of obesity-induced insulin resistance. However, some studies suggest caution when targeting macrophage PTP1B, due to its potential anti-inflammatory role. We assessed the role of macrophage PTP1B in inflammation and whole-body metabolism using myeloid-cell (LysM) PTP1B knockout mice (LysM PTP1B). LysM PTP1B mice were protected against lipopolysaccharide (LPS)-induced endotoxemia and hepatic damage associated with decreased proinflammatory cytokine secretion in vivo. In vitro, LPS-treated LysM PTP1B bone marrow-derived macrophages (BMDMs) displayed increased interleukin (IL)-10 mRNA expression, with a concomitant decrease in TNF-α mRNA levels. These anti-inflammatory effects were associated with increased LPS- and IL-10-induced STAT3 phosphorylation in LysM PTP1B BMDMs. Chronic inflammation induced by high-fat (HF) feeding led to equally beneficial effects of macrophage PTP1B deficiency; LysM PTP1B mice exhibited improved glucose and insulin tolerance, protection against LPS-induced hyperinsulinemia, decreased macrophage infiltration into adipose tissue, and decreased liver damage. HF-fed LysM PTP1B mice had increased basal and LPS-induced IL-10 levels, associated with elevated STAT3 phosphorylation in splenic cells, IL-10 mRNA expression, and expansion of cells expressing myeloid markers. These increased IL-10 levels negatively correlated with circulating insulin and alanine transferase levels. Our studies implicate myeloid PTP1B in negative regulation of STAT3/IL-10-mediated signaling, highlighting its inhibition as a potential anti-inflammatory and antidiabetic target in obesity.

ΔNp63 Promotes Pediatric Neuroblastoma and Osteosarcoma by Regulating Tumor Angiogenesis

The tumor suppressor gene p53 and its family members p63/p73 are critical determinants of tumorigenesis. ΔNp63 is a splice variant of p63, which lacks the N-terminal transactivation domain. It is thought to antagonize p53-, p63-, and p73-dependent translation, thus blocking their tumor suppressor activity. In our studies of the pediatric solid tumors neuroblastoma and osteosarcoma, we find overexpression of ΔNp63; however, there is no correlation of ΔNp63 expression with p53 mutation status. Our data suggest that ΔNp63 itself endows cells with a gain-of-function that leads to malignant transformation, a function independent of any p53 antagonism. Here, we demonstrate that ΔNp63 overexpression, independent of p53, increases secretion of interleukin (IL)-6 and IL-8, leading to elevated phosphorylation of STAT3 (Tyr-705). We show that elevated phosphorylation of STAT3 leads to stabilization of hypoxia-inducible factor 1α (HIF-1α) protein, resulting in VEGF secretion. We also show human clinical data, which suggest a mechanistic role for ΔNp63 in osteosarcoma metastasis. In summary, our studies reveal the mechanism by which ΔNp63, as a master transcription factor, modulates tumor angiogenesis.

Absence of the Shb adaptor protein accelerates leukemia progression by control of cytokine responses

Leukemic progression is highly dependent on cytokine secretion and the cytokine responses within the leukemic cell population. Increased production of hematopoietic cytokines such as G-CSF and IL-6 has for instance been demonstrated as a key element promoting the development of leukemia. Shb is a widely expressed, SH2-domain containing, adaptor protein downstream of active tyrosine kinase receptors that assembles signaling complexes consisting of numerous signaling components of relevance to hematopoietic cell function, of which FAK stands out as particularly important. Absence of Shb was recently shown to reduce hematopoietic stem cell (HSC) proliferation and thus we sought to investigate Shb's role in the progression of leukemia. After transformation of wild type and Shb null bone marrow (BM) cells with a retroviral BRC-Abl construct and subsequent transplantation to wild type recipients it was noted that the Shb knockout BCR-Abl transformed cells produced a more aggressive disease with death occurring at an earlier time point. These mice exhibited an elevated white blood cell count at the time of death and this coincided with an increased relative number of mature neutrophils. In the BM of mice that received the Shb knockout BCR-Abl transformed cells, the c-Kit+Lin-BCR-Abl+ population of cells displays significantly increased proliferation and reduced apoptosis as determined by FACS analysis after staining for Ki-67 and cleaved caspase 3. Cytokine hyper-responsiveness is a characteristic of aggressive leukemias; the Shb knockout BM response to a gradient of GM-CSF was significantly elevated compared to the wild type at lower cytokine concentrations. Moreover, significantly elevated G-CSF mRNA was observed in c-Kit+ cells of the Shb knockout BM after BCR-Abl transformation. Intracellular signaling events were also altered in these cells as demonstrated by elevated phosphorylation of STAT3 and FAK. Further studies are required to determine Shb's exact position in the network that regulates hematopoietic cytokine expression and signaling, but the data implicates Shb as an important modulator of leukemic disease progression with particular relevance to neutrophil dysplasia. Leukemic progression is highly dependent on cytokine secretion and the cytokine responses within the leukemic cell population. Increased production of hematopoietic cytokines such as G-CSF and IL-6 has for instance been demonstrated as a key element promoting the development of leukemia. Shb is a widely expressed, SH2-domain containing, adaptor protein downstream of active tyrosine kinase receptors that assembles signaling complexes consisting of numerous signaling components of relevance to hematopoietic cell function, of which FAK stands out as particularly important. Absence of Shb was recently shown to reduce hematopoietic stem cell (HSC) proliferation and thus we sought to investigate Shb's role in the progression of leukemia. After transformation of wild type and Shb null bone marrow (BM) cells with a retroviral BRC-Abl construct and subsequent transplantation to wild type recipients it was noted that the Shb knockout BCR-Abl transformed cells produced a more aggressive disease with death occurring at an earlier time point. These mice exhibited an elevated white blood cell count at the time of death and this coincided with an increased relative number of mature neutrophils. In the BM of mice that received the Shb knockout BCR-Abl transformed cells, the c-Kit+Lin-BCR-Abl+ population of cells displays significantly increased proliferation and reduced apoptosis as determined by FACS analysis after staining for Ki-67 and cleaved caspase 3. Cytokine hyper-responsiveness is a characteristic of aggressive leukemias; the Shb knockout BM response to a gradient of GM-CSF was significantly elevated compared to the wild type at lower cytokine concentrations. Moreover, significantly elevated G-CSF mRNA was observed in c-Kit+ cells of the Shb knockout BM after BCR-Abl transformation. Intracellular signaling events were also altered in these cells as demonstrated by elevated phosphorylation of STAT3 and FAK. Further studies are required to determine Shb's exact position in the network that regulates hematopoietic cytokine expression and signaling, but the data implicates Shb as an important modulator of leukemic disease progression with particular relevance to neutrophil dysplasia.

Abstract 2308: ΔNp63 promotes neuroblastoma by regulating tumor angiogenesis.

Abstract Background: The tumor suppressor gene p63 and its family members p53/p73 have been described as critical determinants of tumorigenesis. ΔNp63, a splice variant of p63 lacking the N-terminal transactivation domain, is thought to antagonize the transcriptional regulation of the p53, p63 and p73 target genes and blocks their tumor suppressor activity. Overexpression of ΔNp63 has been observed in a number of adult human cancers, suggesting a role of this isoform in tumor formation. While some studies have proposed that ΔNp63 might simply inhibit p53 function within cells, leading to malignant transformation, we found that this model is challenged by the lack of consistent correlation between p53 mutation and ΔNp63 expression suggesting that ΔNp63 might possess a gain of function. Methods: Since expression of ΔNp63 isoforms in primary tumors has been shown to correlate with poor prognosis, we attempted to analyze their relationships with tumor angiogenesis. To test whether overexpression of oncogenic ΔNp63 in HEK-293T cells can stimulate tube formation in endothelial cells using HUVEC tube formation assay. Further, we performed gene affymetrix as well as protein micro array analysis in primary cells compared to cells overexpressed ΔNp63 isoforms. In addition, we used siRNA techniques to verify any potential role of ΔNp63 isoforms in tumor angiogenesis in vitro as well as in vivo experiments. Results: We found that overexpression of the oncogenic isoform of p53 family member ΔNp63 in HEK-293T cells resulted in stimulation of tubular structures in HUVEC cells. As 293T cells lack functional p53, these results strongly suggest a gain of function for ΔNp63. In addition, we found that ΔNp63 is overexpressed in neuroblastoma and ΔNp63 overexpression results in increasing secretion of the inflammatory cytokines interleukin-6 (IL-6) and -8 (IL-8) leading to elevated phosphorylation of STAT-3. Our further analysis showed that elevated phosphorylation of STAT-3 induced stabilization of HIF-1α protein resulting in VEGF secretion. Conclusions: We found that ΔNp63 is overexpressed in neuroblastoma and resulted in elevated secretion of the inflammatory cytokines IL-6 and -8 leading to increased STAT3 activation. Our further studies showed that elevated ΔNp63 induced stabilization of HIF-1α protein resulting in VEGF secretion in a p53 independent manner. Importantly, we show in vitro and in vivo experiments that depletion of ΔNp63 isoform by specific siRNAs significantly reduced angiogenic potential of Neuroblastoma. In summary, our study revealed the underlying molecular mechanism how ΔNp63, as a master transcription factor, modulates tumor angiogenesis in Neuroblastoma. Citation Format: Hemant K. Bid, Maren Cam, Antony Audino, Raushan Kurmasheva, Jiayuh Lin, Peter Houghton, Hakan Cam. ΔNp63 promotes neuroblastoma by regulating tumor angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2308. doi:10.1158/1538-7445.AM2013-2308

Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

BackgroundConstitutive activation of signal transducer and activator of transcription 3 (Stat3) signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer.ResultsWe found that elevated Stat3 phosphorylation in 19 of 100 (19%) bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F) and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC). The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin) and a cell cycle regulating gene (cyclin D1) was associated with the cell growth inhibition and apoptosis.ConclusionThese results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

Stat3 activation in human endometrial and cervical cancers

The activation of signal transducer and activator of transcription 3 (Stat3) has been implicated in the oncogenesis of cancer and is regarded as a novel target for cancer therapy. Stat3 is classified as a proto-oncogene, because an activated form of Stat3 can mediate oncogenic transformation in cultured cells and tumour formation in nude mice. The constitutive activation of Stat3 has been frequently detected in various types of human cancers. However, the constitutive activation of Stat3 in endometrial and cervical cancers has not been studied. We examined tyrosine phosphorylation of Stat3 (activated form of Stat3) in multiple endometrial and cervical cancer tissues using tissue microarray slides as well as cancer cell lines to explore the possible activation of Stat3. Our results indicated that elevated phosphorylation of Stat3 was detected in cervical and endometrial cancer cell lines. Our results also showed that elevated levels of phosphorylation of Stat3 protein were detected in the endometrial and cervical cancer specimens. This is the first study to demonstrate that Stat3 is activated in human endometrial and cervical cancer tissues. Immunohistochemical staining showed that activated Stat3 is associated with increased expression of downstream antiapoptotic genes, Bcl-xL, survivin, and Mcl-1 in these tissues. Expression of a dominant-negative Stat3 mutant using adenovirus-mediated gene transfer inhibited cell growth and induced apoptosis in HeLa and SiHa cervical cancer cell lines expressing elevated levels of Stat3 phosphorylation. Further, a JAK/Stat3 small molecular inhibitor, JSI-124, induced apoptosis more selectively in HeLa and SiHa cancer cell lines than Ishikawa cell line without elevated levels of Stat3 phosphorylation. These results indicate that Stat3 is activated in human endometrial and cervical cancers and the inhibition of constitutive Stat3 signaling may be an effective target for cancer intervention in these two cancers.